The pH-dependent vesicle membrane binding property of chromogranin A appears to be of fundamental physiological importance with regard to the potential roles of chromogranin A in secretory vesicle biogenesis, particularly in segregating secretory vesicle membranes from others in the trans-Golgi network, and also in transmitting extravesicular signals such as inositol 1,4,5-triphosphate or inositol 1,3,4,5-tetrakisphosphate for Ca2+ release or uptake to the inside of vesicles. Several vesicle matrix proteins of the secretory vesicles including chromogranins A and B bound to the vesicle membrane at the intravesicular pH of 5.5 and were freed from it when the pH was raised to a near physiological pH of 7.5. Estimation of the fraction of vesicle matrix proteins that might remain bound to the vesicle membrane in the vesicle suggested that the majority (>50-80%) of chromogranins A and B as well as several other proteins will stay bound to the membrane in the vesicle. Since it has been demonstrated with chromogranin A that the conserved near N-terminal region of chromogranin A exhibited the pH-dependent membrane binding activity, the same region in chromogranin B (residues 17-36) was tested using a synthetic chromogranin B peptide, and found to exhibit the pH-dependent membrane binding activity. The pH-dependent binding of the matrix proteins at pH 5.5 and the automatic untethering at a physiological pH accord well with the rapid release and circulation of the vesicular contents in the bloodstream. Furthermore, the pH-dependent binding of chromogranins to the secretory vesicle membrane implicates essential roles chromogranins might play in the secretory vesicle biogenesis, i.e. protein and membrane sorting processes in the trans-Golgi network.